Quantitative analysis of Oct4 / Xist RNA + DNA templates in single blastomeres and embryos. Individual embryos or blastomeres at the 8-cell stage were processed in a single tube from collection to template quantification. In order to measure both RNA and DNA copy numbers, RT was carried out in all these samples, followed by duplex LATE-PCR. Xist-specific fluorescence (solid lines) is reported on the left scale of each graph, Oct4-specific fluorescence (broken lines) is reported on the right scale of each graph. (A) Comparison of Oct4/Xist RNA + DNA analysis in a female and a male blastomere. The male blastomere contained a single copy of Xist genomic DNA (no Xist RNA), shown by the solid blue line, and a much higher number of Oct4 templates (indicating Oct4 expression) shown by the broken blue line. The single-copy Oct4 signal generated by a polar body is shown for reference (broken green line). The female blastomere also contained numerous Oct4 templates (broken red line) and an even higher number of Xist templates (solid red line) due to active Xist expression. The sex of these blastomeres was confirmed by analysis of the other cells comprising each embryo. (B) Consistency of template measurements in single cells and embryos. The Xist signal of an 8-cell male embryo (solid black line) crossed the threshold three duplication cycles earlier than the Xist signal of one male blastomere (solid blue line), consistent with the presence of eight genomes in the embryo as also confirmed by Xist amplification in 10-genome equivalents of standard DNA (light-green line). In this example, the Oct4 template numbers of the cell and embryo analyzed (blue and black broken lines, respectively) showed a 1:8 ratio similar to that of the Xist templates. This ratio, however, can vary because, while Xist is not expressed in males, Oct4 measurements include RNA levels that differ among blastomeres (see Fig. 5).